Human tissue plasminogen activator has been identified and described as a particularly important and potent new biological pharmaceutical agent that has shown extraordinary results in the treatment of vascular diseases, such as myocardial infarction, due to its high fibrin specificity and potent ability to dissolve blood clots in vivo.
Human tissue plasminogen activator has been the subject of numerous scientific and patent application disclosures. Although its existence prompted numerous investigations by several scientific groups, it was first identified as a substantially pure isolate from a natural source, and tested for requisite plasminogen activator activity in vivo. by Collen et al., European Patent Application Publn. No. 41766, published Dec. 16, 1981, based upon a first filing of June 11, 1980. See also the corresponding scientific publication is Rijken et al., J. Biol. Chem. 256, 7035 (1981).
Subsequently, human tissue plasminogen activator was fully identified and characterized by underlying DNA sequence and deduced amino acid sequence based on successful work employing recombinant DNA technology resulting in large quantities of t-PA in a distinct milieu. This work was recorded in the scientific literature (Pennica et al., Nature 301, 214 (1983)) and in European Patent Application Publn. No. 93619, published Nov. 9, 1983, based upon a first filing of May 5, 1982.
Using the latter disclosure as a basic tool, numerous other researchers have reported on the thus enabled preparation of the molecule via recombinant DNA technology. Certain of these researchers also have publicly disclosed the potential of variants of the basic structure, mentally foreseeing derivatives that may vary in overall biological or pharmacokinetic effects. The resultant public disclosures for the most part have been prophetic and equivocal in terms of actual overall biological or pharmacological results.
Analogous endeavors in the laboratories that succeeded first in producing t-PA recombinantly have been recorded factually in terms of confirmed molecule characterization and observed biological effect, both in the scientific literature and in various patent applications, notably those cited and incorporated herein by reference via the text of the opening paragraph of this application. In all events, the trend seems to favor research along lines of endeavoring to modify the basic structure of human tissue plasminogen activator in order to fully explore and exploit its commercial potential according to various biologically based endpoints.
Based partly upon such research and disclosures, it seems now clear that the human tissue plasminogen activator molecule contains five domains (stretches of amino acid sequence) that have been defined with reference to homologous or otherwise similar structures identified in various other proteins such as trypsin, chymotrypsin, plasminogen, prothrombin, fibronectin and epidermal growth factor. These domains have been designated, starting at the N-terminus of the amino acid sequence of human tissue plasminogen activator, as 1) the finger region (F) that has variously been defined as including amino acid 1 upwards of about 44, 2) the growth factor region (G) that has been variously been defined as stretching from about amino acid 45 upwards of amino acid 91 (based upon its homology with EGF), 3) kringle one (K1) that has been defined as stretching from about amino acid 92 to about 173, 4) kringle two (K2) that has been defined as stretching from about amino acid 180 to about amino acid 261 and 5) the so-called serine protease domain (P) that generally has been defined as stretching from about amino acid 264 to the C-terminal end of the molecule. These domains are situated contiguously generally of one another, or are separated by short "linker" regions, and account for the entire amino acid sequence of from 1 to 527 amino acids in its putative mature form.
Each domain has been described variously as contributing certain specific activity: that is, the finger domain has been variously described as containing a sequence essential or at least of major importance for high binding affinity to fibrin. (This activity is thought important for the high specificity human tissue plasminogen activator displays with respect to clot lysis at the locus of a fibrin rich thrombus.) The growth factor-like region likewise has been associated with cell surface binding activity, at least with respect to urokinase. The Kringle 2 region has also been strongly associated with fibrin binding and with the ability of fibrin to stimulate the activity of t-PA. The serine protease domain seems to enjoy unanimous agreement of being the workhorse domain of the molecule in respect of plasminogen activating activity.
Again, it is noted that the finger region has been generally regarded as spanning amino acids 1.44 of the N-terminus and various researchers have endeavored to produce mutants or variants deleting or partially deleting segments of this domain. Reference is again made to co-pending application Ser. No. 07/068,448, filed June 30, 1987, in this regard.
N-linked glycosylation sites exist in the molecule at amino acid positions 117, 184, 218 and amino acid 448. The site at amino acid 218 is not glycosylated. The glycosylation site at amino acid 117 has been characterized as being a high mannose type, while the other two sites display so-called complex oligosaccharide structures. Sites 117 and 448 seem always to be glycosylated, when the molecule is derived from a host cell capable of effecting glycosylation, while site 184 is thought to be glycosylated in about 50 percent of the molecules. The latter 184 glycosylated/unglycosylated phenomenon has been demonstrated via SDS-PAGE analysis where two bands can be seen, one associated with 184 glycosylated molecules and the other 184 unglycosylated molecules: so-called Type I and Type II t-PA. This partial glycosylation pattern may be the result of site 184 being situated in a conformationally sheltered position between the two kringle structures. For a more detailed discussion of the glycosylation structures of t-PA, reference again is had to co-pending Ser. No. 07/118,098, filed Nov. 6, 1987, and its parents.
A third locus that has received scientific attention is the so-called proteolytic cleavage site within the region defined by amino acids 275 to about 279, and more particularly, the bond between amino acid 275 and 276 of the native molecule. Again, reference is made, in this respect, to co-pending Ser. No. 07/071,506, filed July, 9, 1987, and its parents. Mutagenesis at this site so as to make it less susceptible to proteolytic degradation creates a molecule that remains in a single-, or one-chain, form that is thought to have certain advantages biologically and commercially.
All of these defined domains, glycosylation sites and one-chain/two-chain cleavage site have been described and defined as having specific potential biological activity components. For example, removal of a substantial portion or all of the finger domain results in a molecule with substantially diminished fibrin binding characteristics, albeit in return there is a decrease in the overall rate of clearance of the resultant entity--see Ser. No. 07/068,448.
Modification of the native molecule so as to destroy the one-chain to two-chain cleavage site, as such, results in a molecule with somewhat altered biological activity and more stability while the fibrin binding and fibrin stimulation are increased relative to two-chain t-PA--see Ser. No. 07/071,506.
Alteration of the glycosylation sites, and in particular at amino acid 117, seems invariably to result in a molecule having affected solubility characteristics, that may result additionally in an altered T1/2life pattern and/or fibrin binding characteristics see Ser. No. 07/118,098.
Given that high fibrin specificity and binding characteristics are desirable results to be possessed by human tissue plasminogen activator, and in particular, variously altered derivatives or variants thereof (See, for example, European Patent Application Publication No. 234,051, published Sept. 2, 1987), the art serves to teach away from altering the finger region, dramatically decreased clearance rates--See U.S. Ser. No. 07/068,448, cited supra. And yet, given the commercial significance of fibrin binding and fibrin specificity, it is a perceived goal among researchers to produce variants or derivatives of human plasminogen activator that would have high fibrin binding activity without altering the other desirable biological and pharmacokinetic properties otherwise associated with the native material. However, the research path for producing such variants or derivatives of human plasminogen activator is not altogether clear from the art extant. See, for example, European Patent Application Publication No. 231,624, published Aug. 12, 1987.
The uncertainty as to whether and where to alter the t-PA native molecule for perceived improved fibrinolytic properties is particularly emphasized by a relatively recent patent publication identified as WO 87/04722 (published Aug. 13, 1987). This document reflects an elaborate paper mosaic of potential variants of t-PA. Although the publication refers to three regions, namely the amino N-terminus, glycosylation sites and single chain cleavage site, there is no evidence of actual preparation of t-PA species, and no bioactivity or other data; hence, the publication merely "contemplated" that the proteins possess improved fibrinolytic profiles relative to native human t-PA without specific reference as to what is meant by that, either qualitatively or quantitatively. Indeed, many of the variants arguably generically embraced may have lower fibrinolytic activity. As such, it serves at best as a relatively complex mosaic from which one may be invited to experiment; not more.